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Adult muscle formation requires Drosophila Moleskin for proliferation of
wing disc-associated muscle precursors
Kumar Vishal, David S. Brooks, Simranjot Bawa, Samantha Gameros, Marta Stetsiv, and Erika
R. Geisbrecht
Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS
66506
Genetics: Early Online, published on March 1, 2017 as 10.1534/genetics.116.193813
Copyright 2017.
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RUNNING TITLE
Moleskin and indirect flight muscle formation
KEY WORDS
Drosophila melanogaster, indirect flight muscles, Moleskin, proliferation
CORRESPONDING AUTHOR
Erika R. Geisbrecht, Ph.D., 141 Chalmers Hall, Kansas State University, Manhattan, KS 66506;
(785)532-3105; geisbrechte@ksu.edu
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ABSTRACT
Adult muscle precursor (AMP) cells located in the notum of the larval wing disc undergo rapid
amplification and eventual fusion to generate the Drosophila melanogaster indirect flight muscles
(IFMs). Here we find that loss of Moleskin (Msk) function in these wing disc-associated myoblasts
reduces the overall AMP pool size, resulting in the absence of IFM formation. This myoblast loss
is due to a decrease in the AMP proliferative capacity and is independent of cell death. In contrast,
disruption of Msk during pupal myoblast proliferation does not alter the AMP number, suggesting
that Msk is specifically required for larval AMP proliferation. It has been previously shown that
Wingless (Wg) signaling maintains expression of the Vestigal (Vg) transcription factor in
proliferating myoblasts. However, other factors that influence Wg-mediated myoblast
proliferation are largely unknown. Here we examine the interactions between Msk and the Wg
pathway in regulation of the AMP pool size. We find that a myoblast-specific reduction of Msk
results in the absence of Vg expression and a complete loss of the Wg pathway readout β-
catenin/Armadillo (Arm). Moreover, msk RNAi knockdown abolishes expression of the Wg target
Ladybird (Lbe) in leg disc myoblasts. Collectively, our results provide strong evidence that Msk
acts through the Wg signaling pathway to control myoblast pool size and muscle formation by
regulating Arm stability or nuclear transport.
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INTRODUCTION
Stem cell pool proliferation is critical in the regulation of tissue size and organization in normal
development and mediates repair processes following injury (MICCHELLI and PERRIMON 2006;
GONZALEZ 2007; EGGER et al. 2008; JIANG and EDGAR 2012). The extent of cell proliferation
required to generate different tissues is variable and is generally influenced by the size of an initial
precursor pool balanced by the frequency of cell division and/or cell subsequent differentiation.
For example, a lack of neural stem proliferation during neural circuit formation can result in
microcephaly in mice (HOMEM et al. 2015). Studies performed in both mice and Drosophila show
that intestinal stem cell proliferation dictates tissue maintenance and repair (JIANG and EDGAR
2012). Coordination between cell proliferation and cell differentiation is critical for the formation
and maintenance of larval blood cell generation and ovarian development in Drosophila (GILBOA
2015). Ultimately, common mechanisms unite proliferative processes that form diverse tissues.
A number of evolutionarily conserved signaling pathways are known to regulate stem cell
proliferation. For example, Wingless (Wg)/Wnt signaling is the principle regulator of mammalian
intestinal stem cell proliferation (JIANG and EDGAR 2012). Similarly, Hippo signaling maintains
lung cell homeostasis by controlling the proliferation of epithelial stem cells (LANGE et al. 2015).
Although some factors that regulate stem cell proliferation have been widely studied (BRACK et
al. 2008; TAKASHIMA et al. 2008; BENMIMOUN et al. 2012; CHEN et al. 2012; SHIM et al. 2013) ,
additional components and detailed mechanisms controlling stem cell proliferation are not clearly
understood.
The indirect flight muscles (IFMs) of Drosophila melanogaster serve as a good model
system to understand mechanisms that regulate stem cell proliferation (FERNANDES et al. 1991;
ROY 1999; DUTTA 2006). The IFMs are subdivided into two groups, the dorsoventral muscles
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(DVMs) and the dorsal longitudinal muscles (DLMs). In DLM formation, muscle stem cells called
adult muscle precursors (AMPs) are set aside in embryogenesis and will eventually give rise to
this set of IFMs (BATE et al. 1991; FIGEAC et al. 2007; FIGEAC et al. 2011). The AMPs remain in
an undifferentiated state and undergo rapid rounds of proliferation in the notum region of the wing
disc during the second (L2) and third (L3) larval instar stages to generate ~2500 myoblasts within
a period of 120 hours (h) (GUNAGE et al. 2014). At the onset of pupation, most thoracic larval
muscle fibers undergo histolysis. However, three dorsal oblique muscles are spared and split into
six fibers that serve as DLM templates, also called organizer or founder muscles (ROY and
VIJAYRAGHAVAN 1998; BERNARD et al. 2003). The AMPs undergo an additional round of
proliferation and myoblast fusion to form the eventual six DLM fibers that are approximately one-
third of their final size. These muscles increase in volume for the remaining three days of pupal
development and each DLM ends end up with ~3000 myonuclei (KOPEC 1923). Thus, the rapid
proliferation of muscle stem cells during larval and pupal development is critical for proper IFM
formation.
A network of transcription factors are responsible for the massive proliferative increase in
wing disc-associated AMPs. Twist (Twi) and Notch are required for maintaining myoblasts in a
proliferative phase to block muscle differentiation (BATE et al. 1991; ANANT et al. 1998; BERNARD
et al. 2010). Disrupting Notch function in the AMPs downregulates Twi expression and results in
premature differentiation (ANANT et al. 1998). In contrast, Mef2 is the major differentiation factor
that promotes IFM formation and is activated by the anti-differentiation protein Twi
(RANGANAYAKULU et al. 1995; CRIPPS and OLSON 1998; BRYANTSEV et al. 2012). This paradox
is resolved by findings that Twi and Notch activate the Holes in muscle (Him) transcription factor
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to prevent premature muscle differentiation through suppression of Mef2 activity (LIOTTA et al.
2007; SOLER and TAYLOR 2009)
The transcriptional activity required for the large increase in the larval AMP pool size is
developmentally regulated. Notch pathway activation is initiated in embryogenesis and extends
into larval development to promote AMP amplification, while soluble Wg protein released from
the disc epithelial cells in L3 larvae supercedes Notch signaling (GUNAGE et al. 2014). Wg then
acts through Arm and Pangolin/T-cell factor (TCF) upstream of the transcription factor Vestigial
(Vg) to control the myoblast precursor pool and subsequent IFM formation (SUDARSAN et al.
2001). In support of this, expression of dominant negative TCF in myoblasts decreases Vg protein
expression, reduces the AMP pool size and impairs IFM formation (SUDARSAN et al. 2001). The
wing disc-associated myoblasts that express Vg and low levels of another transctipiton factor
called Cut generate the IFMs, while myoblasts that express high levels of Cut give rise to direct
flight muscles (DFMs). Thus, Vg and Cut act in a mutually repressive manner during muscle
formation to generate distinct muscle types.
How the Wg and Notch signaling pathways integrate with Vg and other wing disc-
associated proteins in the amplification of AMPs is still unclear. Here we demonstrate a new role
for Drosophila Moleskin (Msk)/Importin-7 (Dim7) in regulation of the adult myoblast pool size.
Msk is a member of the Importin-β superfamily of proteins and is involved in nuclear protein
transport in both vertebrates and invertebrates (GÖRLICH et al. 1997; MASON and GOLDFARB
2009). Studies in vertebrate skeletal myogenesis demonstrate that Importin-7 is required for the
nucleo-cytoplasmic shuttling of Extracellular signal-related kinase (ERK) to regulate myoblast
proliferation and differentiation (MICHAILOVICI et al. 2014). Similarly, Drosophila Importin-7
regulates the nuclear transport of ERK and in turn influences cell proliferation in wing disc
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development (MARENDA et al. 2006). However, Msk also has functions independent of its nuclear
shuttling role during embryonic myogenesis. An Elmo-Msk protein complex localizes to the sites
of myotendinous junction (MTJ) formation and msk mutants exhibit muscle detachment
phenotypes (LIU and GEISBRECHT 2011; LIU et al. 2013).
In this paper, we highlight a new role for Msk in the regulation of muscle stem cell numbers
during Drosophila adult muscle formation. We find that blocking Msk function in the wing disc-
associated muscle precursors results in a drastic reduction in the overall size of the AMP pool and
leads to the absence of DLMs. This lack of muscle formation is a result of impaired larval AMP
amplification as disruption of Msk function in pupal myoblast proliferation does not affect the
myoblast pool size and has a minor effect on DLM formation.
MATERIALS AND METHODS
Fly strains and Genetics
All fly lines used in this study were grown on standard cornmeal medium at 25 °C unless
otherwise stated. The following fly strains were obtained from published sources: da-Gal4 (for
qPCR; from Mitch Dushay); 1151-Gal4 (myoblast-specific driver from L.S. Shashidhara) and
rp298-Gal4 (founder cell-specific driver from Susan Abmayr). The following fly strains were
obtained from the Bloomington stock center: UAS-nls-GFP [w[1118]; P{w[+mC]=UAS-
GFP.nls}14 (BL4775)]; UAS-GFP RNAi [w[1118]; P{w[+mC]=UAS-GFP.dsRNA.R}143
(BL9331)]; UAS-DN-TCF [y[1] w[1118]; P{w[+mC]=UAS-pan.dTCFDeltaN}4 (BL4784)],
UAS-armS10 [P{UAS-arm.S10}C, y1 w1118 (BL4782)]; UAS-msk full length (FL) [w[*];
P{w[+mC]=UAS-msk.L}47M1/CyO (BL23944)]; UAS-sgg(WT) [w[1118]; P{w[+mC]=UAS-
sgg.B}MB5 (BL5361)]; UAS-sgg(Y241F) [w[1118]; P{w[+mC]=UAS-sgg.Y214F}2 (BL6817)];
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and two UAS-msk RNAi lines [ y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02727}attP2 (BL-
27572)] and [y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS00020}attP2 (BL33626)] (LIU et al.
2013). The following fly stocks were generated in the lab using standard genetic crosses: 1151-
Gal4; UAS-Gal80ts (for temporal regulation of transgene expression); UAS-DN-TCF; UAS-msk
FL (for rescue of DN-TCF); UAS-GFP, UAS-msk RNAi (control for armS10 rescue); UAS-
armS10, UAS-msk RNAi (for rescue of armS10); UAS-msk FL; UAS-sgg (WT) (for rescue of
sgg); and UAS-msk FL; UAS-sgg (Y214F) (for rescue of activated sgg).
Immunostaining
Appropriately staged wandering L3 larvae were selected to analyze the role of Msk in regulating
muscle stem cell number in wing and leg imaginal discs. Wing discs from individual larvae were
dissected and fixed in 4% formaldehyde for 25 minutes (min) at room temperature followed by
immunohistochemistry. To examine the effect of Msk function in adult IFM muscle formation, 0
h pupae (white pupae) were collected and aged to specific time points: 20 h after puparium
formation (APF) (splitting of larval template/organizer cell is completed) or 24 h APF (initial steps
of DLM patterning is completed). Pupal preparations were dissected, fixed and immunostained.
The myoblast pool in both wing and leg discs were labeled using rabbit anti-Twist (Twi; 1:300;
Krzystof Jagla), mouse anti-Ladybird (Lbe; 1:1000, Krzystof Jagla); guinea pig anti-Earthbound
1 (Ebd; 1:4000; Yashi Ahmed); mouse anti-Cut [1:100; Developmental Studies Hybridoma Bank
(DSHB)], rabbit anti-Vestigal (Vg; 1:100; Sean Carroll), and rabbit anti-Mef2 (1:100; Bruce
Patterson). Cell death was analyzed using Acridine orange (AO) staining (Sigma) and TUNEL
assays (Roche) using standard protocols (MILÁN et al. 1997). Muscle differentiation was examined
using mouse anti-Myosin heavy chain (MHC; 1:500; Susan Abmayr). Myoblast proliferation was
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monitored using Rabbit anti-phospho-Histone 3 (PH3; 1:100; Millipore). Mouse anti-β-
Catenin/Arm (1:100; DSHB) was analyzed as a readout of Wg signaling. Myonuclei were labelled
using rabbit anti-Erect wing (Ewg; 1:2000, Yashi Ahmed) and developing fibers were monitored
using mouse anti-22C10 (1:100 DSHB). Secondary antibodies for fluorescent immunostaining
were Alexa Fluor 488 and Alexa 546. Immunostained preparations were imaged on a Zeiss 700
confocal microscope and images were processed using Zeiss ZEN software and Photoshop
Elements 12.
Quantitative PCR to verify Msk knockdown by RNAi
Three samples of five larvae each were prepared for the da-Gal4/+ and da-Gal4>msk RNAi
genotypes. Many da-Gal4>msk RNAi larvae died before reaching the L3 stage, so five L2 larvae
were homogenized in the buffer provided in the kit. Three RNA samples for each phenotype were
used for analysis. RNA was generated using the RNeasy Mini Kit (Qiagen). After elution, RNA
concentrations were determined using Thermo Scientific’s Nanodrop 1000. Single strand cDNA
was generated from 150ng RNA using the SuperScript III First-Strand Synthesis System Kit
(Invitrogen). For the qPCR reactions, each cDNA solution was diluted 1:50 and mixed with SYBR
Select Master Mix for CFX (Applied Biosystems). rp49 was used as the reference gene. Primers
for the qPCR reactions were synthesized by Integrated DNA Technologies (IDT):
msk: Left – TTGCGCGCAACTATTGATCC, Right – CTTGAGGTAGACAGCACCGG
rp49: Left – GCCCAAGGGTATCGACAACA, Right – GCGCTTGTTCGATCCGTAAC
Reactions were run in triplicate using the BioRad CFX96 Touch™ Real-Time PCR Detection
System with CFX Manager Software. The average of the triplicates was used to calculate the 2^-
ΔΔCT values (Normalized fold expression).
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Quantitation and Statistical Analysis
The size of the myoblast pool was quantitated using two different methods: (1) Myoblast density
was determined by counting the total number of Twi(+), Mef2(+), or Ebd(+) nuclei in three random
regions (1600 µm2 area each) of the wing imaginal disc-associated myoblasts; and/or (2) by
counting the total number of wing disc-associated Twi(+) myoblasts in single nodal planes of 1µm
thickness. Myoblast proliferation was calculated by determining the percentage of PH3(+)/Cut(+)
notum myoblasts. Wg signaling was monitored by comparing the fraction of Twi(+) or Mef2(+)
myoblasts that co-localized with Arm nuclei in a single nodal plane. N ≥ 15 individuals for each
genotype in each experiment. Fiber formation in the pupal stages was analyzed by comparing the
total number of 22C10(+) developing fibers per hemisegment between the control and
experimental animals at specific time points. IFM myonuclei were monitored by counting the total
number of Ewg(+) nuclei per fiber (MUKHERJEE et al. 2011). N = 6-8 individuals for each genotype
were quantified.
Myoblast quantifications were performed using the ‘Analyze Particles’ function in ImageJ,
recorded in an Excel spreadsheet, and imported into Graphpad Prism 6.0 software for the
generation of graphs and statistical analysis. The column statistics function was used to confirm
statistically significant sample sizes and normality. All error bars represent the mean ± S.E.M.
Statistical significances were determined using either student t-tests, Mann-Whitney tests, or one-
way ANOVA followed by a Bonferroni post‐hoc analysis. Differences were considered significant
if P < 0.05 and are indicated in each figure legend. All reagents used in this study are available
upon request.
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RESULTS
Msk is required in the larval AMPs for IFM formation.
Our laboratory previously found that msk is essential for the establishment of muscle-
tendon attachment in Drosophila embryogenesis (LIU and GEISBRECHT 2011; LIU et al. 2013). As
an extension of this work, we sought to evaluate the contribution of Msk in adult myogenesis. We
and others have found that msk mutants are lethal prior to pupal stages, thus precluding analysis
of adult IFM formation (LORENZEN et al. 2001; LIU and GEISBRECHT 2011; LIU et al. 2013;
NATALIZIO and MATERA 2013). To circumvent this limitation and to examine the role of Msk in
the pupal stages of IFM development, we utilized the binary Gal4/UAS expression system to
knockdown Msk using RNAi. First, we confirmed that two independent UAS-msk RNAi lines
effectively reduced msk transcript levels using quantitative PCR (qPCR) (Fig. S1). The stronger
UAS-msk RNAiHMS is used for the remainder of our studies. Knockdown of Msk was further
confirmed in this line using newly generated antisera against the Msk protein (Fig. S1).
To examine whether a reduction in Msk affects the pool of actively dividing myoblasts that
give rise to the IFMs, knockdown of Msk was accomplished using 1151-Gal4. Analysis of GFP
fused to the SV40 nuclear localization signal (UAS-nls-GFP) confirms expression of the 1151-
Gal4 driver in proliferating adult myoblasts located in the notum region of the L3 wing disc (Fig.
1A,B; yellow box) (FERNANDES et al. 2005). Control discs of 1151-Gal4 alone possess
approximately 2500 myoblasts by the end of the L3 stage and can be visualized using the myoblast
markers Twi (Figs. 1C,F) or Mef2 (Fig. 1I). Knockdown of msk transcript in these same
proliferating myoblasts substantially reduced the myoblast pool (Figs. 1G,J). While the position
of the remaining myoblasts within the notum varied, the reduction in the overall myoblast pool
size remained constant among different samples (Figs. 1H,K). Note that basal expression of the
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UAS-msk RNAi line with no driver (Fig. S1) or knockdown of GFP as a negative control
(1151>GFP RNAi) (Fig. 1D,E) did not alter the number of myoblasts.
The msk RNAi-induced reduction in myoblast density suggests that Msk may be required
for cell death, muscle differentiation and/or cell proliferation in the AMPs. To examine if apoptosis
could be a cause of the decreased myoblast pool, we examined 1151-Gal4 control and 1151>msk
RNAi notum myoblasts for activated Caspase 3 or AO positive cells. We failed to detect any
indication of cell death in the AMPs (Fig. S2). Next we immunostained L3 wing discs with an
antibody against Myosin heavy chain (MHC) which detects differentiated muscle tissue (LOVATO
et al. 2005). No staining was observed in control or msk RNAi myoblasts (Fig. S3), indicating that
premature muscle differentiation was not a cause of AMP reduction. The marker phospho-Histone
3 (PH3) is specific for cells undergoing mitosis. On average, a small fraction of Cut-labeled
myoblasts also stain for PH3 (Fig. 1L,N). A decrease in Msk reduced the fraction of PH3(+)/Cut(+)
myoblasts (Fig. 1M,N). Thus, here we conclude that Msk is required for myoblast proliferation in
the notum region of L3 wing discs.
The large increase in proliferating myoblasts during the larval stages ensures a sizeable
myoblast pool during fusion to generate the adult IFMs. By 12 h APF, larval muscle histolysis is
complete and myoblasts migrate towards three persistent DLM muscle templates for fusion and
muscle growth. Rapid myoblast fusion continues between 12-18 h APF and induces splitting of
the three larval scaffolds into six DLM fibers (WEITKUNAT and SCHNORRER 2014). Myoblast
nuclei labeled with anti-Ewg antisera are present in the developing DLMs marked with 1151-
driven GFP expression at 20 h APF (Fig. 2A) and 24 h APF (Fig. 2F). This data is consistent with
previous reports where 1151 is observed in the developing IFM fibers in pupal development
(ANANT et al. 1998; DUTTA et al. 2004). This continued expression of the 1151 promoter in
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proliferating and fusing myoblasts allowed us to test the effect of persistent msk RNAi knockdown
during pupal DLM development. Control preparations of 1151-Gal4 alone or 1151>GFP RNAi
both showed the full complement of six IFM fibers at 20 h APF (Fig. 2B,C,E; asterisks) and 24 h
APF (Fig. 2G,H,J; asterisks). Reduction of msk during proliferation of the larval AMPs
dramatically decreased the number of muscle fibers analyzed at both 20 h (Figs. 2D,E) and 24 h
APF (Figs. 2I,J). Notably, the remaining muscles appear to be larval templates as the Ewg-stained
nuclei are larger than the nuclei present in developing DLM fibers. Together, these results suggest
that Msk is required to produce a minimal number of myoblasts sufficient for fiber size and/or
splitting. Failure to maintain this proliferative state may cause muscle degeneration since we never
observed three larval templates in 1151-driven msk RNAi individuals.
To further determine if Msk is required in all myoblasts or specifically affects a subset of
myoblasts, msk RNAi was expressed under control of the duf/kirre promoter (rp298-Gal4). rp298
is expressed in the three larval templates that serve as founder muscles in organizing future DLM
fiber development during myoblast fusion (FERNANDES et al. 1991; DUTTA et al. 2004). A subset
of wing disc-associated myoblasts express GFP under control of the rp298 promoter (Fig. 3A).
Compared to rp298-Gal4 (Fig. 3B) or rp298>GFP RNAi (Fig. 3C) controls, fewer myoblasts were
observed after a reduction in msk RNAi levels (Fig. 3D). Accordingly, this reduction in myoblast
number (Fig. 3E) also resulted in decreased fiber number. The six DLMs normally present at 20 h
APF (Figs. 3F-H; asterisks) or 24 h APF (Fig. 3K-M; asterisks) were decreased to approximately
four fibers upon induction of msk RNAi by rp298-Gal4 (Figs. 3I,J; asterisks) at 24 h APF (Figs.
3N,O; asterisks). These data implicate Msk as an important player in the generation and/or
maintenance of the myoblast pool, both in founder cells and fusing myoblasts.
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Our results thus far show that a reduction in Msk activity was initiated during larval AMP
proliferation and persisted through pupal myoblast proliferation, myoblast fusion, and fiber
splitting. While the final number of DLM fibers was decreased upon msk RNAi by 24 h APF (Figs.
2J; 3O), we could not distinguish between a requirement for Msk in generation of the larval or
pupal myoblast pool. Therefore, we utilized the Gal4/Gal80ts TARGET system to bypass the
requirement for Msk during larval development and determine if Msk is essential for pupal
myoblast proliferation (BRAND and PERRIMON 1993; SUDARSAN et al. 2001; MCGUIRE et al.
2004). Gal80ts is a temperature-sensitive (ts) mutation that binds to and inactivates the Gal4 protein
at permissive temperatures (18 °C). This inactivation prevents the Gal4 expression of UAS-driven
elements. At restrictive temperatures (29 °C), the Gal80ts protein loses its ability to repress Gal4
and allows for the induction of UAS transgenes. 1151-Gal4; UAS-Gal80ts control pupae at 24 h
APF possessed six DLM fibers (Fig. 4B,C; asterisks). Individuals expressing msk RNAi were
shifted to the non-permissive temperature of 29 °C at 0 h APF, which corresponds to the beginning
of pupal development (Fig. 4A). Dissection at 24 h APF yielded a small, yet significant reduction
in fiber number after a decrease in Msk function (Fig. 4F,G; asterisks). This reduction is likely
caused by a delay in fiber splitting (Fig. 4G; arrow) and not due to a lack of muscle formation from
defective pupal myoblast proliferation or aberrant fusion as the number of myoblasts observed in
control or msk RNAi knockdown muscle fibers were similar (Fig. 4D,E,H,I).
Msk influences Wg signaling
IFM formation requires Vg, a transcriptional activator that is expressed at higher levels in
the proximal myoblasts of the wing disc (Fig. 5A,B; yellow asterisks) (SUDARSAN et al. 2001;
BERNARD et al. 2003). In contrast, the Cut transcription factor is normally highest in myoblasts
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closest to the wing hinge (Fig. 5A,C; white arrows) and gives rise to the DFMs. Vg staining is
absent from the entire notum myoblast pool upon msk RNAi knockdown (Fig. 5D,E), while Cut
protein persists in the remaining myoblasts (Figs. 5D,F). Thus, loss of Msk affects the presence of
Vg, a key regulator of IFM development. Since Vg represses Notch in developing DLMs
(BERNARD et al. 2006), we examined whether loss of Vg via msk RNAi altered Notch signaling.
However, the levels or subcellular localization of the Notch intracellular domain (NICD) were not
altered in control or msk RNAi wing discs (Fig. S4), suggesting that Msk does not affect activated
Notch in notum myoblasts.
Vg is a known transcriptional target of Wg signaling in the developing wing pouch
(SWARUP and VERHEYEN 2012) and may be a direct target of Wg signaling in the AMPs
(SUDARSAN et al. 2001). Few Wg targets have been identified in wing disc notum myoblasts. Thus,
we wondered if additional Wg-regulated genes in other cell types also require Msk function in
other populations of adult myoblasts. Maqbool, et al. identified Ladybird (Lbe) as a target of
extrinsic Wg signaling in the developing leg disc (MAQBOOL et al. 2006). Lbe(+) myoblasts were
observed in both dorsal and central regions of the leg disc and partially colocalized with the
myoblast marker Earthbound 1 (Ebd) (Fig. 5G-I; yellow asterisks). Msk reduction by RNAi
abolished leg disc myoblast expression of Lbe (Fig. 5J-L). These results suggest a broader role for
Msk in general myoblast proliferation and maintenance of the Wg-responsive proteins Vg and Lbe
in different tissues.
We next sought to place Msk within the Wg signaling pathway. Ebd is a DNA binding
protein that physically interacts with the transcriptional co-activators Arm and TCF in context-
specific Wg-dependent processes, including IFM formation (BENCHABANE et al. 2011; XIN et al.
2011). Ebd is present in L3 wing disc-associated AMPs (Fig. 6A). While there is a sharp decrease
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in the number of Ebd(+) myoblasts upon loss of Msk (Fig. 6C), the expression of Ebd was not
altered (Fig. 6B). Next we examined the relationship between Msk and the Wg co-activator TCF.
Overexpression of full length Msk (Msk FL) did not affect the myoblast pool (compare Fig. 6E to
1151-Gal4 controls in Fig. 1E,H,K). Consistent with published results, expression of dominant-
negative TCF (DN-TCF) in the AMPs using the 1151-Gal4 driver reduced the overall myoblast
number (Fig. 6E,G) (SUDARSAN et al. 2001). However, the introduction of excess Msk in a DN-
TCF background did not alter the size of the AMP pool (Fig. 6F,G), indicating that Msk does not
act downstream of TCF.
To test the hypothesis that Msk functions upstream of Wg-responsive transcription, we
next examined whether an activated version of Armadillo (armS10) could rescue the myoblast
deficit resulting from msk RNAi knockdown. Expression of armS10 alone in myoblasts had no
effect on the overall myoblast pool (Fig. 6H). However, the introduction of activated Arm in a msk
RNAi background (Fig. 6K) partially rescued the total number of myoblasts compared to msk RNAi
alone (Fig. 6I). In contrast, expression of UAS-driven GFP did not alter the number of myoblasts
compared to msk RNAi alone (Fig. 6J), indicating that an additional UAS-line did not dilute out
the effectiveness of the Gal4 protein. Quantitation using two different parameters confirmed
rescue. Counting both myoblast density (Fig. 6L) and the total number of myoblasts in a single
plane (Fig. 6M) revealed an increase when armS10 was expressed in a msk RNAi background over
msk RNAi alone or msk RNAi, UAS-GFP. Here we conclude that Msk lies upstream of the
Arm/TCF transcriptional complex, although these experiments cannot rule out the role of Msk in
a parallel pathway.
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Proper Msk function is essential for Arm in the myoblast pool
The subcellular localization of Arm plays a pivotal role in transducing Wg signaling. The
kinase Shaggy (Sgg)/GSK-3β phosphorylates cytoplasmic Arm and targets the protein for
destruction by the proteasome in the absence of Wg ligand (BEJSOVEC 2006; SWARUP and
VERHEYEN 2012). However, in cells that receive Wg signal, Sgg and other members that comprise
this so-called destruction complex are inactivated, resulting in cytoplasmic Arm accumulation and
translocation to the nucleus to activate Wg-responsive genes. If Msk acts upstream of Arm, we
may expect to see altered subcellular localization of the Arm protein.
A percentage of myoblasts expressing Arm was observed as a readout of Wg signaling in
controls (Fig. 7A-C; D, arrowheads). The location of these myoblasts appeared stochastic as
individual wing-discs showed different populations of Arm(+) cells within the proliferating
myoblast population. However, abrogation of Msk function nearly eliminated Arm expression in
the remaining myoblasts (Fig. 7E-G). Quantitation showed a reduction in Arm(+)/Twi(+) cells
from about 20% in control myoblasts to less than 2% upon decreased Msk function (Fig. 7H).
To further examine the relationship between Wg and Msk, we analyzed the distribution of
Twi-expressing myoblasts relative to the source of Wg ligand in the notum epithelial cells.
Consistent with previous results (SUDARSAN et al. 2001), Wg protein was detected in a stripe of
cells underlying the myoblast pool (Fig. 7I-K). This is further demonstrated by XZ scans showing
myoblasts in a plane above the Wg-expressing epithelial cells (Fig. 7L). Analysis of msk RNAi
wing discs revealed a number of insights. First, Wg protein distribution was not altered upon msk
RNAi knockdown (Fig. 7M,N). This important observation suggests that Msk does not act in a cell
non-autonomous manner to influence Wg production and/or secretion. Second, the remaining
myoblasts in XY plane views were still present at the most dorsal and ventral regions of the wing
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disc (Fig. 7O,P; white arrows), suggesting that Msk responds to Wg signaling at distant locations
from ligand production. Finally, msk-depleted myoblasts were also observed in a layer most distal
from the site of Wg production (Fig. 7P, yellow arrow). We conclude from these experiments that
there is no correlation between the source of Wg and ability of Msk to respond to Wg signaling to
promote myoblast proliferation.
The cytoplasmic to nuclear translocation of Arm is usually a readout of Wg signaling.
Surprisingly, Arm protein was undetectable in msk RNAi wing disc-associated myoblasts. To test
the hypothesis that Msk may be required for Arm protein stability, we chose to block Wg signaling
in the L3 AMPs by overexpressing either WT [UAS-sgg (WT)] or an activated version of Sgg
[UAS-sgg (Y214F)] (BOUROUIS 2002). Excess sgg (WT) significantly decreased the overall
myoblast density (Fig. 8B,D) compared to 1151 controls (Fig. 8A,D). Note that this block in Wg
signaling was comparable to the overexpression of DN-TCF in AMPs (Fig. 6E,G). Overexpression
of the activated sgg (Y241F) line, which is thought to retain low levels of kinase activity, also
showed a decrease in myoblast density (Fig. 8F,H). This disruption in Wg signaling was less severe
compared to the expression of sgg (WT), but significant compared to 1151 controls (Fig. 8E,H).
Next we tested if overexpression of Msk altered the Sgg-dependent reduced myoblast number.
Indeed, driving Msk FL in larval AMPs either partially or fully restored the myoblast pool in a Sgg
(WT) (Fig. C,D) or Sgg (Y214F) (Fig. 8G,H) background. Note that expression of Msk alone did
not alter myoblast number (Fig. 6D,G; 8D,H). These data, taken together, suggest Msk may affect
the Sgg destruction complex upstream of the Arm/TCF co-activator proteins responsible for Wg
transcriptional responses.
19
DISCUSSION
Drosophila makes two sets of muscles during its life cycle: embryonic body wall muscles required
for larval movement and adult muscles necessary for flight, climbing, and mating. An important
difference between these two muscle sets is their size. Embryonic muscles are smaller and are
comprised of ~ 2-35 myonuclei (BATE 1990). This final muscle size is largely dependent on the
total number of fusion events that occur in myogenesis. In contrast, the muscles that comprise one
subset of adult muscles, the IFMs, are large and each muscle fiber is made up of ~ 3000 myonuclei.
The small 8-12 cell AMP precursor pool that generates these muscles is set aside in the embryo,
remain undifferentiated, and become associated with wing imaginal discs (BATE et al. 1991).
Rapid proliferation of these AMPs in the later larval and early pupal stages is critical for attaining
final muscle cell size. Taken together, these results suggest that IFM myogenesis provides us a
unique opportunity to understand factors that regulate AMP pool size and in turn muscle formation.
In this study, we demonstrate a new role for Msk in the regulation of AMP number and
DLM formation. First, reduced Msk function results in minimal fiber formation, likely a
consequence of decreased AMP numbers. In theory, this smaller pool size could be due to reduced
proliferation, increased cell death or premature muscle differentiation. Two possibilities have been
ruled out: increased cell death was not observed in the myoblast pool upon Msk reduction and no
indication of premature muscle differentiation was present in msk RNAi animals. However,
blocking Msk function resulted in a severe depression in myoblast proliferation as assayed by PH3
staining. Thus, our data shows that the reduction in myoblasts is largely due to reduced AMP
amplification during the normally proliferative larval stages.
Our lab and others have obtained evidence that IFM formation is dictated by the size of the
AMP pool. Disruption of TCF or overexpression of the Vg-repressor Cut results in a severe
20
depletion in AMP number and minimal DLM fiber development (SUDARSAN et al. 2001). Our data
also shows that blocking Msk function in all myoblasts causes a strong reduction in the overall
size of the myoblast pool and a lack of fiber formation. However, the effect on the AMP population
and DLM number is less severe when Msk function is reduced only in founder myoblasts.
Similarly, blocking Rac1 GTPase activity in notum myoblasts causes only a minor reduction in
AMP number and a slight reduction in fiber number (FERNANDES et al. 2005). This strong
correlation between cell proliferation and organ size has been an emerging theme in multiple
development systems (BREUNINGER and LENHARD 2010; TUMANENG et al. 2012).
Msk is a member of the β-like importin family of proteins and is most similar to
mammalian Importin-7 (53% identity and 71% similarity to mouse Imp-7). The canonical role of
the β-like importin family is in the nuclear import of proteins in response to extracellular stimuli
(FLORES and SEGER 2013). This import can be dependent or independent of cargo containing a
classical nuclear localization signal (NLS) and sometimes requires a physical interaction with
importin-β proteins. A number of cargoes have been identified that require Imp-7 for nuclear
translocation. Some are general cellular proteins, such as ribosomal proteins (JÄKEL et al. 1999;
FASSATI et al. 2003; FREEDMAN and YAMAMOTO 2004), while others are transcription factors that
are imported in response to stimulation, including both vertebrate and Drosophila ERK (LORENZEN
et al. 2001; MICHAILOVICI et al. 2014). However, we did not observe an effect of Msk reduction
on dpERK subcellular localization or protein levels in the myoblasts (Fig. S4). Moreover, RNAi
knockdown of the Drosophila Importin β homolog Ketel, which is required for the nuclear import
of dpERK in embryos (LORENZEN et al. 2001), did not reduce the AMP pool in notum myoblasts
(Fig. S5). Together, our results suggest that Msk and Ketel do not function together in AMP
21
proliferation, but highlight the importance of a novel role for Msk in response to external Wg as a
signaling stimulus.
Where does Msk fit within the known paradigm of Drosophila AMP proliferation and IFM
formation? A study by Gunage, et al., shows that the initial post-embryonic amplification of AMPs
in L2 larvae is regulated by the Notch pathway and further AMP proliferation is under control of
Wg signaling in L3 larvae (GUNAGE et al. 2014). Furthermore, secreted Wg acts through TCF in
the AMPs for the maintenance of Vg expression in myoblast proliferation and subsequent IFM
muscle formation (SUDARSAN et al. 2001). Since our data shows that Vg protein is absent upon a
reduction in Msk function, we reasoned that Msk may be responsive to Wg signaling. The
introduction of excess Msk does not rescue decreased myoblast proliferation due to a block in Wg
signaling by DN-TCF. However, activated Arm partially rescues the myoblast deficit resulting
from msk RNAi. Collectively, these results place Msk upstream or parallel to TCF and Arm to
regulate myoblast pool size through Vg.
Our findings suggest that Msk may regulate the myoblast pool size by two possible
mechanisms, which are not mutually exclusive. First, Msk may directly regulate the nuclear import
of Arm and/or TCF in response to Wg stimulation. A second possibility is that Msk may control
the stability of Arm in the cytoplasm. Excess levels or activation of Sgg, one of the components
of the Arm destruction complex (Apc/Axin/Sgg) reduces AMP pool size. This result suggests that
Arm stability is critical for myoblast amplification. Interestingly, overexpressing Msk in
combination with excess or activated Sgg partially rescues the myoblast pool. Taken together, our
results support the idea that Msk might regulate myoblast pool size by controlling Arm stability
through Sgg. Our future experiments will be aimed at examining if Msk biochemically interacts
with destruction complex proteins.
22
Does Msk regulate Wg signaling that controls myoblast pool size in other muscle groups?
Leg muscles are derived from a population of myoblasts associated with imaginal leg discs. One
of the known Wg targets, Lbe, is expressed widely in leg disc myoblast and is known to regulate
muscle growth and performance (MAQBOOL et al. 2006). Loss of Wg signaling results in the loss
of Lbe expression in the leg disc myoblasts, which in turn leads to impaired muscle patterning.
Our data shows that Msk dictates myoblast pool size in the leg disc. Similar to Wingless loss of
function, blocking Msk results in the absence of Wg target Lbe in the leg disc myoblasts.
Collectivity, these results suggest that Msk might function as a general regulator of Wg signaling
during muscle formation.
The data here increases our general understanding of stem cell regulation, subsequent organ
formation, and patterning during development. Furthermore, since AMPs phenocopy some
features of vertebrate satellite cells, our findings may provide insight into mechanisms regulating
satellite cell proliferation following muscle injury in vertebrates. Our next step is to examine
whether Msk regulates the amplification of myoblast pool size during muscle injury or aging.
ACKNOWLEDGMENTS AND FUNDING
We are grateful to Susan Abmayr and Mitch Dushay for Drosophila stocks and to Krzystof
Jagla, Yashi Ahmed, Sean Carol, and Bruce Patterson for sharing antibodies. We would also like
to thank the Integrated Genomics Facility in the Department of Plant Pathology at Kansas State
University for assistance with the qPCR results. Stocks obtained from the Bloomington Drosophila
Stock Center (NIH P40OD018537) were used in this study. Monoclonal antibodies from the
Developmental Studies Hybridoma Bank (DHSB) were created by the NICHD of the NIH and are
maintained at The University of Iowa for monoclonal antibodies. We also thank Nicole Green for
23
reading the manuscript and her valuable comments. This work was supported by the National
Institutes of Health (RO1AR060788 to E.R.G.).
FIGURE LEGENDS
Figure 1. Msk is required for the generation of the wing disc-associated myoblast pool. (A,B)
The 1151-Gal4 driver is used to express nls-GFP in all larval wing disc-associated myoblasts at
the L3 stage. (A) Low magnification of the wing disc (white dotted outline). The yellow boxed
region shows the location of the larval myoblasts in the notum (B). (C,D,F,G,I,J) Maximum
projection confocal microscopy images of the AMP pool in control (C,D,F,I) or 1151>msk RNAi
(G,J) L3 wing discs labeled with the myoblast markers Twi (C,D,F,G) or Mef2 (I,J). Note that the
myoblast pool (dotted line) is reduced upon disruption of Msk (G,J) compared to controls (C,D,F).
(E,H,K). Quantitation of myoblast density (per regions 1600 µm2) in control (1151-Gal4 or
1151>GFP RNAi) and msk RNAi (1151>msk RNAi) wing discs labeled with Twi (E,H) or Mef2
(K). (L-N) PH3 staining to monitor proliferating notum myoblasts. More Cut(+) myoblasts also
stain for PH3 in control (L) compared to msk RNAi (M) discs. (N) Bar graph showing the fraction
of PH3(+)/Cut(+) myoblasts. Mean +/- S.E.M. (****, p < 0.001; ***, p < 0.005; n.s., not
significant). Scale bar: 50µm.
Figure 2. Abrogated Msk function during larval myoblast proliferation reduces DLM fiber
number. (A-D,F-I) Maximum projection confocal micrographs of DLM fibers at 20 h APF (A-D)
or 24 h APF (F-I). (A,F) Pupal myoblasts labeled with Ewg (red) are being incorporated into the
developing DLM fibers (asterisks) marked by 1151-driven GFP (green) at 20 h APF (A) or 24 h
APF (F) through reiterative myoblast fusion events. (B-D,G-I) Developing DLMs are stained with
24
22C10 (green) to mark muscle fibers and Ewg (red) to label myoblasts. (B,C,G,H) 1151-Gal4
(B,G) or 1151>GFP RNAi control (C,H) animals have six DLM fibers (asterisks) at 20 h APF
(B,C) or 24 h APF (G,H). (D,I) Little fiber formation is seen in 1151>msk RNAi animals. (E,J)
Quantitation of fiber number shows there are significantly fewer DLM fibers upon knockdown
with msk RNAi animals compared to controls. Mean +/- S.E.M. (****, p < 0.001; n.s., not
significant). Scale bar: 50µm.
Figure 3. Blocking Msk function in founder cells reduces both the myoblast pool size and
fiber number. (A-D) Maximum projection confocal pictures of L3 wing disc-associated
myoblasts. (A) rp298 expression is present in a subset of myoblasts as visualized by GFP
expression. (B-D) The numbers of larval AMPs labeled with anti-Twi is similar in rp298-Gal4 (B)
or rp298>GFP RNAi (C) controls, but reduced in rp298>msk RNAi expressing myoblasts (D).
Dotted lines denote larval myoblast pool. (E) A bar graph showing a significant reduction in the
density of myoblasts (per regions 1600 µm2) present in the notum of rp298>msk RNAi wing discs
compared to controls. (F-O) The consequences of msk RNAi knockdown in DLM fibers at 20 h
APF (F-J) or 24 h APF (K-O). Fibers are marked by 22C10 (green; asterisks) and fused myonuclei
are labeled with Ewg (red). Six fibers are present in controls (G,H,L,M), whereas rp298>msk RNAi
individuals have less than six fibers (I,N). (J,O) Bar graphs showing significantly fewer fibers per
hemisegment at 20 h APF (J) or 24 h APF (O). Mean +/- S.E.M. (****, p < 0.001; **, p < 0.01; *,
p < 0.05; n.s., not significant). Scale bar: 50µm.
Figure 4. Knockdown of msk RNAi during pupal morphogenesis does not alter the number
of myonuclei, but causes a minor delay in fiber formation. (A) Schematic showing the
25
temperature shift paradigm for msk RNAi induction during pupal development. (B-D,F-H)
Maximum projection confocal micrographs of DLM fiber formation at 24 h APF. Fibers are
marked by 22C10 (red; asterisks) and fused myonuclei are immunostained with Ewg (green). (B-
D) Controls have the normal complement of six fibers. (F-H) A mild decrease in fiber number is
observed upon msk RNAi knockdown. Note that complete fiber splitting is delayed (G, white
arrow). (E) Bar graph quantitates the small decrease in fiber number upon a reduction in Msk. (I)
Quantitation reveals no difference in the number of myonuclei between control (D) and
experimental samples (H). Mean +/- S.E.M. (***, p < 0.005; n.s. = not significant). Scale bar:
50µm.
Figure 5. Msk regulates the expression of Wg-responsive genes in the wing imaginal disc and
leg disc. (A-F) Myoblasts in L3 wing discs immunolabeled with Cut (red) and Vg (green) in
controls (A-C), compared to those with disrupted Msk function (1151>msk RNAi) (D-F). While
both Vg and Cut exhibit broad myoblast expression, Vg (A,B; *) accumulates at higher levels in
the dorsal myoblasts while Cut (A,C; arrows) protein is seen at increased levels in ventral
myoblasts. Cut staining (D,F) is still present, while Vg expression is absent (D,E) upon induction
of msk RNAi. (G-L) Effect of blocking Msk function on Lbe expression in leg disc-associated
myoblasts. In control animals (G-I), myoblasts are double labelled with Ebd (green) and Lbe (red,
asterisks). (J-L) Disruption of Msk function results in a significantly fewer Ebd(+) myoblasts
accompanied by loss of Lbe expression. All images are Z-stack projections. Scale bar: 50µm.
26
Figure 6. Moleskin acts upstream of Wg transcriptional complexes.
(A,B) Maximum projection confocal images of wing disc-associated myoblasts marked by Ebd
antibody in control and msk RNAi samples. (C) The myoblast density (per regions 1600 µm2) is
significantly less in msk RNAi (B) samples compared to controls (A). (D-G) Effect of
overexpressing Msk in a dominant-negative TCF mutant background. Myoblasts are marked by
Twi in the notum region of L3 wing discs in Z-stack projections. (D) Overexpression of Msk alone
does not alter the myoblast pool number. (E) Expression of DN-TCF results in reduced density of
the myoblast pool. (F) Overexpression of Msk in a DN-TCF background does not rescue the
reduction in myoblast number. (G) Quantification of the myoblast pool density in the indicated
genotypes. (H-K) Effect of overexpressing armS10 in an msk RNAi mutant background in
maximum intensity projections. (H) The Twi-labeled myoblast pool in armS10 wing discs is
similar to controls. (I, J) A diminished myoblast pool is present in both 1151>msk RNAi (I) and
1151>GFP; msk RNAi (J) wing discs. (K) Overexpressing armS10 partially rescues the myoblast
pool size. (L, M) Quantitation comparing the myoblast density (L, per regions 1600 µm2) or
myoblast pool size per single confocal plane (M) in the indicated genotypes. Mean +/- S.E.M.
(****, p < 0.001; ***, p < 0.005; **, p < 0.01; n.s. = not significant). Scale bar: 50µm.
Figure 7. Disrupting Msk function results in loss of Arm protein. (A-G) Immunofluorescent
double-labeling of Arm (green) and myoblasts (red) marked by Twi antibody in L3 wing discs.
(A-C) Controls show an accumulation of Arm in a fraction of myoblasts depicted as maximum
intensity projections. (D) Single plane orthogonal views through the notum wing disc showing co-
localization of Twi(+) and Arm(+) myoblasts. The yellow arrow points from the epithelium
towards the myoblast layers in the notum. (E-G) There is no Arm accumulation in the remaining
myoblasts in 1151>msk RNAi wing discs shown as Z-stack projections. (H) Bar graph shows a
27
significant reduction in the fraction of Arm(+) myoblasts upon a reduction of Msk. (I-K) Maximum
projection confocal images of L3 wing discs double labeled with Wg (green) and Twi (red). (L) A
single plane orthogonal section of the wing disc showing Wg-expressing epidermal cells and the
overlying myoblasts. The yellow arrow points away from the source of Wg towards the myoblasts.
(M-O) Similar to controls, the myoblast pool (red) in msk RNAi animals is evenly distributed
relative to the Wg(+) (green) cells. Also, there is no difference in Wg staining between the control
and the experimental samples. (P) Orthogonal section of notum wing discs show that the myoblasts
are juxtaposed next to a source of Wg, but maintain their location at the distal edge of the myoblast
layer. The yellow arrow points away from the source of Wg towards the myoblasts. Note that two
different samples are shown in (M) and (P). In all orthogonal views, the upper panel corresponds
to an XZ view of the red line and the green line is the location of the XZ view of the green line.
Mean +/- S.E.M. (****, p < 0.001). Scale bar: 50µm.
Figure 8. Msk acts through Sgg to regulate the wing disc myoblast pool size.
(A-C, E-G) Maximum confocal projections of L3 notum myoblasts immunostained with Twi. (A-
C) (A-B) Qualitatively, less myoblasts are present upon overexpression of wild-type sgg (WT) (B)
compared to 1151 controls (A). (C) Overexpressing Msk in a sgg (WT) background partially
rescues the myoblast number. (D) Quantification of myoblast density (per regions 1600 µm2) in
panels A-C. (E,F) Targeting a weak version of activated sgg (214F) (F) causes a reduction in the
myoblast pool compared to 1151 controls (E). (G) A significant increase in the myoblast pool size
is seen in 1151>msk FL;sgg (Y214F) samples. (H) A bar graph showing partial restoration of the
myoblast pool (per regions 1600 µm2) upon overexpression of msk FL in a sgg (Y214F)
28
background. Mean +/- S.E.M. (****, p < 0.001 ***, p < 0.005, n.s. = not significant). Scale bar:
50µm.
29
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